1. Field of the Invention
The present invention relates to a novel molecule involved in the process of apoptosis. More specifically, isolated nucleic acid molecules are provided encoding the human RAIDD protein and two splice variants thereofxe2x80x94referred to as RAIDD-SV1 and RAIDD-SV2. RAIDD polypeptides are also provided, as are vectors, host cells and recombinant methods for producing the same. The invention further relates to screening methods for identifying agonists and antagonists of RAIDD activity.
2. Related Art
Apoptosis, or programmed cell death, is a physiological process essential to the normal development and homeostasis of multicellular organisms (Steller, Science 267:1445-1449 (1995)). Derangements of apoptosis contribute to the pathogenesis of several human diseases including cancer, neurodegenerative disorders, and acquired immune deficiency syndrome (Thompson, Science 267:1456-1462 (1995)).
Apoptotic cell death is a multi-phase process (Vaux and Strasser, Proc. Natl. Acad. Sci. USA 93:2239-2244 (1996); Takahashi and Earnshaw, Curr. Opin. Genet. Dev. 6:50-55 (1996)). In the first phase cells react to either external or internal stimuli capable of inducing apoptosis followed by transduction of the stimulus to the cell death effector machinery. In the next phase, cell death machinery is activated resulting in the cells undergoing dramatic changes in structure. This last phase is characterized by the activation of both nucleases and proteases.
Two cell surface receptors, CD95 (Fas/APO-1) and TNFR-1, have been shown to trigger apoptosis by their natural ligands or specific agonist antibodies (Baglioni, Tumor Necrosis Factors, xe2x80x9cThe Molecules and their Emerging Role in Medicine,xe2x80x9d B. Beutler, ed., pp. 425-438 (1992); Itoh et al., Cell 66:233-43 (1991); Trauth et al., Science 245:301-05 (1989)). Both death receptors are members of the tumor necrosis factor (TNF)/nerve growth factor (NGF) receptor family which also include TNFR-2, low-affinity MGFR, CD40 and CD30, among others (Smith et al., Science 248:1019-23 (1990); Tewari et al., Cell 81:801-809 (1995)).
CD95 and TNFR-1 share a region of homology, appropriately designed the xe2x80x9cdeath domain,xe2x80x9d required to signal apoptosis (Itoh et al., J. Biol. Chem. 268:10932-7 (1993); Tartaglia et al., Cell 74:845-53 (1993)). This shared xe2x80x9cdeath domainxe2x80x9d suggests that both receptors interact with a related set of signal transducing molecules that, until recently, remained unidentified. Using the two-hybrid system, three death domain-containing molecules, TRADD, FADD/MORT1 and RIP, were isolated (Boldin et al., J. Biol. Chem. 270:7795-8 (1995); Chinnaiyan et al., Cell 81:505-12 (1995); Cleveland et al., Cell 81:479-82 (1995); Hsu et al., Cell 81:495-504 (1995); Stanger et al., Cell 81:513-23 (1995)). Subsequent studies showed that endogenous FADD associates with CD95 in an activation-dependent fashion (Kischkel et al., EMBO 14:5579-88 (1995)), while similarly, endogenous TRADD and RIP were found complexed to activated TNFR-1 (Hsu et al., Cell 84:299-308 (1996); Hsu et al., Immunity 4:387-96 (1996)). It has been postulated that TRADD acts as an adaptor molecule for TNFR-1 (Hsu et al., Cell 84:299-308 (1996)), mediating the interaction of TNFR-1 with FADD, while, by contrast, RIP may be involved in NF-kB signaling (Hsu et al., Immunity 4:387-96 (1996)). A dominant negative version of FADD (FADD-DN) blocks TNF- and CD95-induced apoptosis, suggesting that FADD functions as the common signaling conduit for cytokine-mediated cell death (Chinnaiyan et al., J. Biol. Chem. 271:4961-65 (1996); Hsu et al., Cell 84:299-308 (1996)).
Overexpression of several proteins containing death domains has been shown to result in the induction of apoptotic cell death in the absence of an apoptotic induction stimulus (Vaux and Strasser, supra). Further, a number of viruses have been found to encode specific inhibitors of apoptosis, suggesting a role for apoptosis in antiviral defense.
Overexpression of TRADD induces both apoptosis and NF-kB activation-two of the most important activities signaled by TNFR-1 (Hsu et al., Cell 81:495-504 (1995)). Upon oligomerization of TNFR-1 by trimeric TNF, TRADD is recruited to the receptor signaling complex (Hsu et al., Cell 84:299-308 (1996)). TRADD can then recruit the following signal transducing molecules: 1) TRAF2, a TNFR-2- and CD40-associated molecule (Rothe et al., Cell 78:681-92 (1994); Rothe et al., Science 269:1424-1427 (1995)), that mediates NF-kB activation, 2) RIP, originally identified as a Fas/APO-1-interacting protein by two-hybrid analysis (Stanger et al., Cell 81:513-23 (1995)), that mediates NF-kB activation and apoptosis (Hsu et al., Immunity 4:387-96 (1996)), and 3) FADD, a Fas/APO-1-associated molecule, that mediates apoptosis (Chinnaiyan et al., Cell 81:505-12 (1995); M. P. Boldin et al., J. Biol. Chem. 270:7795-8 (1995); F. C. Kischkel et al., EMBO 14:5579-5588 (1995)).
Studies have demonstrated that FADD can recruit the ICE/CED-3-like protease FLICE to the Fas/APO-1 death inducing signaling complex (Muzio et al., Cell 85:817-827 (1996); Boldin et al., Cell 85:803-815 (1996)).
The first evidence for the involvement of ICE-like proteases in CD95- and TNFR-1 signaling came with the discovery that the poxvirus gene product CrmA blocks cell death triggered by both receptors (Enari et al., Nature 375:78-81 (1995); Los et al., Nature 375:81-3 (1995); Tewari et al., J. Biol. Chem. 270:3255-60 (1995)). In vitro, the serpin CrmA interacts only with the active forms of ICE and ICE-like proteases (Ray et al., Cell 69:597-604 (1992); Tewari et al., Cell 81:801-09 (1995)). Yama and ICE-LAP3, two of the ICE-like enzymes most related to CED-3, are expressed as zymogens that are proteolytically activated upon ligation of CD95 or TNFR-1 (Chinnaiyan et al., J. Biol. Chem. 271:4961-65 (1996); Duan et al., J. Biol. Chem. 271:35013-35 (1996)). However, both Yama and ICE-LAP3 remained a proenzymes in anti-CD95 treated CrmA-expressing cells, suggesting that CrmA inhibits an ICE-like protease upstream of Yama and ICE-LAP3 (Chinnaiyan et al., J. Biol. Chem. 271:4961-65 (1996)).
Phylogenetic analysis of the ICE/ced-3 gene family revealed three subfamilies (Chinnaiyan et al., Current Biology 6:555-62 (1996); Duan et al., J. Biol. Chem. 271:35013-35 (1996)). Yama, ICE-LAP3, and Mch2 are closely related to C. elegans Ced-3 and comprise the Ced-3 subfamily. ICE and the ICE-related genes, ICE rel II, and ICE rel III form the ICE subfamily, while ICH-1 and its mouse homologue, NEDD-2 form the NEDD-2 subfamily. Based on similarities with the structural prototype interleukin-1b converting enzyme, ICE/Ced-3 family members are synthesized as zymogens that are capable of being processed to form active heterodimeric enzymes (Thornberry et al., Nature 356:768-74 (1992)).
The present invention provides isolated nucleic acid molecules comprising a polynucleotide encoding the RAIDD polypeptide having the amino acid sequence shown in FIG. 1 (SEQ ID NO:2) or the amino acid sequence encoded by the cDNA clone deposited as ATCC Deposit Number 97824 on Dec. 13, 1996.
The present invention further provides isolated nucleic acid molecules comprising a polynucleotide encoding RAIDD polypeptide splice variantsxe2x80x94RAIDD-SV1 and RAIDD-SV2.
The present invention also relates to recombinant vectors, which include the isolated nucleic acid molecules of the present invention, and to host cells containing the recombinant vectors, as well as to methods of making such vectors and host cells and for using them for production of RAIDD polypeptides or peptides by recombinant techniques.
The invention further provides an isolated RAIDD polypeptide, and splice variants thereof, having an amino acid sequence encoded by a polynucleotide described herein.
The present invention also provides a screening method for identifying agonists and antagonists of RAIDD induced cell death, which involves contacting cells which express the RAIDD protein with a candidate molecule and comparing the level of RAIDD induced cell death to a standard level, the standard being assayed when contact is made in absence of the candidate molecule; whereby, an increase in RAIDD induced cell death in comparison to the standard indicates that the compound is an agonist and a decrease in RAIDD induced cell death in comparison to the standard indicates that the compound is an antagonist.
The present invention further provides a screening method for identifying compounds capable of interaction with the RAIDD polypeptides of the present invention, which involves contacting a RAIDD polypeptide with a candidate molecule and measuring whether the candidate molecule interacts with the RAIDD polypeptide.